Transgenic dunaliella salina as a bioreactor

ABSTRACT

Disclosed is a method for making a bioreactor comprising a foreign target gene, special selectable markers and  Dunaliella Salina  as host. It is prepared by the genetic transformation techniques that include introducing a foreign target gene into the cells of  Dunaliella Salina  and screening the transformed cells of  Dunaliella Salina . The bioreactor of the present invention can be used as a safe and cheap production system for proteins of pharmaceutical interest including vaccines, especially oral products, in a large scale, because the cells of  Dunaliella Salina  are easy of genetic manipulation in preparation of the bioreactor, nontoxic and edible for humans and animals, and harmless to the environment.

FIELD OF THE INVENTION

[0001] The present invention relates to a bioreactor, preparation anduse thereof, more particularly, this invention relates to a transgenicDunaliella Salina bioreactor, preparation and use thereof.

BACKGROUND OF THE INVENTION

[0002] It is well known that vaccines administered to humans and animalscan induce their immune systems to produce antibodies against manypathologic organisms, such as viruses and bacteria. For example,vaccines containing the given antigens, such as hepatitis B surfaceantigen (HBsAg), human measles virus antigen, malaria antigen,foot-and-mouth disease virus antigen, rabies virus antigen, etc., areimportant measures to prevent and control such infectious diseases ashepatitis B, measles, malaria, foot-and-mouth disease and rabies inhumans or animals. The traditional vaccines are produced from killed orlive attenuated pathogens. Recently, several academic and industriallaboratories have begun experimenting with transgenic microorganisms,plants or animals as novel manufacturing systems. However, theapplications of these methods are limited greatly for their intrinsicdefects which follow: a) some microorganism expression systems lackpost-translational modifications, for example, protein glycosylation toeukaryotic proteins. Some insoluble aggregates, for which to bere-dissolved is very difficult, often emerge during fermentation ofEscherichia coli. In addition, a huge investment of equipments is oftenrequired for the fermentation; b) a high cost is required for culturinganimal cells, and what is more serious is that the recombinant proteinsproduced by transgenic animals may be contaminated with pathogenicviruses from animals, which may be a potential danger to humans. Atpresent, for example, the hepatitis B vaccine used clinically consistsof major protein particles of HBsAg expressed mainly by Saccharomycescerevisiae. Although the vaccine is effective and plays an importantrole in controlling transmission of hepatitis B virus, it still has manyshortcomings, such as low or no reactivity to some people, immune escapeafter inoculation, and high price which prevents itself from usingwidely in the developing countries. Hence, the development of a moreeffective, and cheaper hepatitis B vaccine is significant forworld-widely controlling hepatitis B virus infection.

[0003] As compared with other production systems such as microorganismfermentation and transgenic animals, plant as a bioreactor for producingpharmaceutical proteins is safer and cheaper due to the followingreasons: firstly, plant viruses do not infect humans, so it is safer;secondly, neither expensive culture materials nor complicated equipmentsare needed for culture of plant cells; finally, strictly asepticproduction conditions and a cold storage can be omitted duringdistributing.

[0004] The biomedical studies on transgenic plants have been made since1990's. For example, the expression of HBsAg in tobacco was successfullydemonstrated in 1992, and later many studies were carried out on someedible plants including tomato, lupine, lettuce, etc. The studiesmentioned above have shown that these plants can express human hepatitisB surface antigen protein, but the yield of protein is low. These plantsare not often eaten in raw due to seasonal growth thereof. Therefore, itis difficult for them to be adopted widely among the population,especially children in which hepatitis B virus infection is a majorhealth problem.

[0005] Hence, it is an objective of the present invention to provide anew and effective transgenic Dunaliella salina bioreactor, which can beused to produce many valuable pharmaceutical proteins including vaccinesfor humans and/or animals.

[0006] It is another objective of the present invention to provide amethod for preparing the transgenic Dunaliella Salina bioreactor whichis used to produce pharmaceutical proteins, vaccines and hormonessuitable for humans, animals and plants.

[0007] Unless indicated otherwise, the term “Dunaliella Salina” refersto a kind of organism classified as Chlorophyta; Chlorophyceae;Volvocales; Dunaliellaceae. Dunaliella Salina is, in the shape of pearor ellipse with volume of 50˜1000 μm³, the most halotolerent unicellulareukaryotic organism known. The cell of Dunaliella Salina has two longapical flagella that propel it through the water. The significantdifference between Dunaliella Salina and other green unicellular algaeis that the cell of Dunaliella Salina lacks a rigid cell wall, and isenclosed by a thin layer of elastic plasma membrane. In the cell, thereis the single, large, cup-shaped chloroplast with a pyrenoid. The redeyespot is located at the front end of the cell. Reproduction ofDunaliella Salina thereof includes asexual reproduction in longitudinalsplit as the main manner and sexual reproduction in isogamy.

[0008] The term “transgene”, as used herein, refers to a foreigndouble-stranded deoxyribonucleic acid (DNA) fragment introduced intoDunaliella Salina as host, which can be either extrachromosomal orintegrated into the host genome, and the resulting transgenic DunaliellaSalina can be propagated by normal breeding or not.

[0009] The term “foreign target gene” refers to the DNA sequenceintroduced into the host Dunaliella Salina, which encodes a protein orpeptide product.

[0010] The term “bioreactor” refers to the transgenic animals, plants ormicroorganism, which can be used to produce the proteins or peptidesencoded by the introduced foreign genes.

[0011] The term “selectable marker” refers to the DNA sequence encodingspecial protein or peptide products, and the host carrying such asequence can grow, propagate and be screened in a special selectivemedium.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012]FIG. 1 shows a schematic diagram of the chloroplast expressionvector p64-TNF-MD containing tumor necrosis factor (TNF).

[0013]FIG. 2 illustrates the construction of the Dunaliella Salinaexpression plasmid pCAMBIA-CtxB-SS1 containing recombinant HBsAg.

[0014]FIG. 3 is a schematic diagram of the Dunaliella Salina expressionvector pCAMBIA-DS1644.

[0015]FIG. 4 is a schematic diagram of the Dunaliella Salina expressionplasmid pCAMBIA-CtxB-SS1 containing recombinant HBsAg.

SUMMARY OF THE INVENTION

[0016] On the basis of the findings from repeated and detailed studiesin our laboratory, the present inventors have developed a new andeffective Dunaliella Salina bioreactor that can be prepared byintroducing foreign target genes derived from humans, animals, plants ormicroorganisms into the cells of Dunaliella Salina using the well-knowngenetic transformation techniques, including electroporation,polyethylene glycol (PEG) and gene-gun, and then screening thetransformed cells. The bioreactor can be used to produce many cheapdrugs, vaccines, phytohormones and other bioactive materials for humansor animals, therefore, can fulfill the purposes of the presentinvention.

[0017] The present invention provides a transgenic Dunaliella Salinabioreactor comprising a Dunaliella Salina as host, a foreign target geneand a selectable marker.

[0018] In one preferred embodiment of the present invention, the saidforeign target gene is derived from at least one selected from the groupconsisting of humans, animals, plants or microorganisms.

[0019] In a further embodiment of the present invention, the saidselectable marker is at least one selected from the group consisting ofspectinomycin or streptomycin resistance encoded by aadA gene,chloromycetin resistance encoded by cat gene, kanamycin or neomycinresistance encoded by nptII or neo gene, hygromycin resistance encodedby hyg gene and PPT resistance encoded by Bar gene.

[0020] Further, the present invention also provides a method forpreparing the transgenic Dunaliella Salina bioreactor, which comprises:

[0021] a) Introducing foreign target genes into the cells of DunaliellaSalina with the transformation techniques;

[0022] b) Screening the transformed cells of Dunaliella Salina.

[0023] The present invention further provides the uses of the transgenicDunaliella Salina bioreactor in production of the vaccines for humansand/or animals.

DETAILED DESCRIPTION OF THE INVENTION

[0024] The present invention provides a transgenic Dunaliella Salinabioreactor comprising a foreign target gene, a selectable marker and aDunaliella Salina as a host.

[0025] The use of Dunaliella Salina as the host, as in the presentinvention, is considered to have significant advantages due to thefollowing reasons: Dunaliella Salina is probably the most halotolerantunicellular eukaryote known, and can grow in high salinity environments,such as oceans or brine lakes, etc. The cell of Dunaliella Salina has nocell wall, which permits rapid volume changes in adaptation toextracellular changes in osmotic pressure. As a result, its adaptabilityto the environments is extremely strong, and can live in a variety ofsalt concentration from as low as 0.2% to salt saturation concentration(35%). Dunaliella Salina has a strong capability of photosynthesis andcan synthesize many organic molecules like proteins from water, carbondioxide and inorganic salts under sunlight. For this reason it is easyand cheap to culture the cells of Dunaliella Salina.

[0026] As in one preferred embodiment of the present invention, the saidforeign target gene is derived from at least one selected from the groupconsisting of humans, animals, plants or microorganisms, which can beeither cloned from any genomes from above-mentioned organisms,artificially synthesized or amplified by polymerase chain reaction (PCR)in vitro.

[0027] The examples of the said foreign target gene derived frommicroorganisms and plants includes but not limited to HbsAg, measlesvirus antigen, foot-and-mouth disease virus antigen, rabies virusantigen, larvacide, cytokinin, endochitinase, glucose-amylase P,thaumatin, seed-stored protein genes, and the like.

[0028] The examples of the said foreign target genes derived from humansand animals include but not limited to angiostatin, endostatin,hemoglobin, human factor III, human erythropoietin, interferon, obeseprotein, human interleukin, human granulocyte colony stimulating factor,human macrophage colony stimulating factor, streptokinase, human proteinkinase, growth hormone, tissue plasminogen activator, defensin, tumornecrosis factor, epidermal growth factor, bovine chymosin, antibioticpeptide genes, and the like.

[0029] All of these foreign target genes listed above can be usedindividually to construct at least one expression vector from the groupconsisting of Dunaliella Salina genome expression vectors, chloroplastexpression vectors, and autonomously replicating expression vectors.

[0030] As in a further preferred embodiment of the present invention,the said selectable marker is at least one selected from the groupconsisting of spectinomycin or streptomycin resistance encoded by aadAgene, chloromycetin resistance encoded by cat gene, kanamycin orneomycin resistance encoded by npt II or neo gene, hygromycin resistanceencoded by hyg gene and PPT resistance encoded by Bar gene.

[0031] Further, the present invention provides a method for preparingthe transgenic Dunaliella Salina bioreactor, which comprises:

[0032] a) introducing foreign target genes into the cells of DunaliellaSalina using the transformation techniques.

[0033] b) screening transformants of Dunaliella Salina.

[0034] As noted above, the said transformation techniques in step a) canbe any one of the methods for genetic transformation consisting ofbiological, physical or chemical methods. Specifically, the foreigntarget gene can be introduced into the cells of Dunaliella Salina byeither biological, physical or chemical methods for genetictransformation techniques, and then be expressed in the transformedcells of Dunaliella Salina.

[0035] The said biological method can be such a method that foreigntarget genes are introduced into the cells of Dunaliella Salina byeither agrobacterium Ti plasmid transformation system or plant virusvector system, and then expressed in the transformed cells of DunaliellaSalina.

[0036] The said physical and chemical methods can be one or more of thefollowing methods:

[0037] a. introducing foreign target genes into the cells of DunaliellaSalina by PEG treatment and expressing them;

[0038] b. introducting foreign target genes into the cells of DunaliellaSalina by liposome and expressing them;

[0039] c. introducing foreign target genes into the cells of DunaliellaSalina by electroporation and expressing them;

[0040] d. introducing foreign target genes into the cells of DunaliellaSalina by ultrasonic delivery and expressing them;

[0041] e. introducing foreign target genes into the cells of DunaliellaSalina by gene gun and expressing them;

[0042] f. introducing foreign target genes into the cells of DunaliellaSalina by microinjection and expressing them;

[0043] g. introducing foreign target genes into the cells of DunaliellaSalina by ultraviolet laser microbeam and expressing them;

[0044] h. introducing foreign target genes into the cells of DunaliellaSalina by glass bead agitation and expressing them;

[0045] i. introducing foreign target genes into the cells of DunaliellaSalina by aerosol gene delivery and expressing them;

[0046] According to one preferred embodiment of the present invention,the process used in the invention further comprises a procedure ofconstructing Dunaliella Salina expression vector and a procedure ofculturing Dunaliella Salina before introducing foreign target genes intoDunaliella Salina cells.

[0047] In another preferred embodiment of the present invention, themethod for preparing the transgenic Dunaliella Salina bioreactor of thepresent invention comprises the following steps which were illustratedwith an example of the transgenic Dunaliella Salina expressing HBsAg.

[0048] a) constructing the CtxB-SS1 fusion gene encoding recombinantHbsAg;

[0049] b).constructing the Dunaliella Salina expression vectorpCAMBIA-CtxB-SS1 containing the said fusion gene in step a);

[0050] c) introducing the said expression vector in step b) into thecells of Dunaliella Salina;

[0051] d) selective subculture;

[0052] wherein the said recombinant HBsAg-encoding gene includes all orpart of the sequences of S, PreS1 and PreS2 genes of hepatitis B virus,and combinations thereof; SS1 gene herein refers to the fusion ofsequences of S and PreS1 genes; the CtxB-SS1 fusion gene herein refersto the fusion of cholera toxin B subunit (CtxB) and SS1 genes.

[0053] In one preferred embodiment of the present invention, theCtxB-SS1 fusion gene was inserted between the Dunaliella Salina Hsp70B5′ promoter and the T-Nos terminator to construct an intact expressioncassette for transcription of the CtxB-SS1 fusion gene under the controlof the Dunaliella Salina Hsp70B 5′ promoter.

[0054] In a further preferred embodiment of the present invention, thesaid expression vector pCAMBIA-CtxB-SS1 contained two sequences ofmatrix attachment regions (MAR), MAR1 and MAR2, in the same orientation.The expression cassette of Nit1 5′-Nit1-T-Nos expressing nitratereductase was inserted between MAR1 and MAR2 to screen the transformedDunaliella Salina.

[0055] The present invention also provides the uses of the bioreactor inproduction of vaccines for humans or animals. The transgenic DunaliellaSalina bioreactor of the present invention can be used to prepare orproduce human or animal vaccines containing the following antigens, suchas HBsAg, influenza hemagglutinin, malaria antigen, measles virusantigen, rabies virus antigen, foot-and-mouth disease virus antigen,phytohormones, and the like.

[0056] It is believed that, as compared with other transgenicbioreactors, the transgenic Dunaliella Salina bioreactor of the presentinvention possesses many advantages:

[0057] 1. Dunaliella Salina is a unicellular organism and its proteincontent is higher. The alga propagates very fast and grows withoutseasonal restriction, and it can grow to dense populations in a shortertime, which allows it easy to screen the transformed Dunaliella Salina.

[0058] 2. Dunaliella Salina is a lower eukaryotic green alga lacking ofcell wall, therefore genetic manipulation thereof is easier. Inaddition, there is the single, large, cup-shaped chloroplast in thecell, which is useful for the chloroplast transformation of foreigngenes. The chloroplast transformation is harmless to the environmentsand does not induce gene silencing.

[0059] 3. The cultivation of Dunaliella Salina is based on autotrophicgrowth in media containing inorganic salts, e.g. nitrate as the nitrogensource and carbon dioxide as the exclusive carbon source, so the cost ofthe culture is low. Dunaliella Salina can grow in the high salinitymedia which other organisms hardly live in. Thus, the cost of productioncan be decreased greatly because two models of cultivation, intensiveand extensive, have been used in the large-scale cultivation ofDunaliella Salina.

[0060] 4. Dunaliella salina has the eukaryotic post-translationalprocessing for proteins, such as the formation of disulfide bond andglycosylation, which simplifies processing of the downstream of geneengineering and assures bioactivity and quality of the protein products.

[0061] 5. Dunaliella Salina is, because of being nontoxic and abundantin natural vitamins and polyunsaturated fatty acids, valuable ediblealga. Oral vaccines or drugs produced with Dunaliella Salina can betaken directly even without purification, so that the cost of productionmay be reduced markedly.

[0062] 6. The bioreactor system according to the present invention maybe used to produce drugs or vaccines for humans and/or animals,phytohormones and other bioactive materials.

[0063] Further description is given with the examples which follow, andthese examples are provided for the purposes of illustration and are notintended to limit the scope of the present invention.

EXAMPLES Example 1

[0064] I. Culture of Dunaliella Salina

[0065] The strain and culture conditions are as follows.

[0066] 1.Liquid culture: Dunaliella Salina UTEX1644 was obtained fromThe Culture Collection of Algae at The University of Texas at Austin,and inoculated into Mclachlan culture fluid. In liquid culture, thecells of Dunaliella Salina UTEX1644 were cultured at temperature of20-30° C. in the flasks and under 14:10-hour light-dark cycle with 3000lux.

[0067] 2. Solid culture: Agar was added into Mclachlan culture fluid upto 0.5-0.8% to prepare solid media. The alga cells being isolated fromthe liquid culture were transferred immediately onto the solid mediaprepared above by use of aseptic technique, and then cultured under thesame conditions as liquid culture.

[0068] II. Construction of the chloroplast expression vector p64C-TNF-MDof TNF

[0069] 1. The Vector pSK-atpX contains cloned chloroplast atpA 5′promoter sequence and rbcL 3′ terminator sequence. Therefore, the saidinserted foreign gene can be expressed highly in the chloroplast. Theplasmid pUC19-TNF contains a cDNA fragment of human tumor necrosisfactor (referred to as TNF-α hereinafter), which is about 600 bp inlength and has a BamH I site at one end and a Xba I site at the otherend. The cDNA fragment of TNF-α was inserted between the atpA5′ promotersequence and the rbcL 3′ terminator sequence after the vector pSK-atpXand the plasmid pUC19-TNF were digested with BamH I and Xba I, and thenan intermediate vector pSK-atpK-TNF containing the expression cassetteof human TNF-α gene (atpA5′-TNF-rbcL3′) was constructed.

[0070] 2.The vector p64C contains cloned homologous fragment to thechloroplast of Dunaliella Salina, clpP-trnl-petB, as well as the 5′promoter and 3′ terminator sequences of the chlL gene. Both the vectorp64C and the intermediate vector pSK-atpX-TNF prepared in step 1 weredigested with EcoR V and Sac I, and then the obtained expressioncassette of TNF-α gene was inserted into vector p64C to construct thechloroplast intermediate expression vector p64C-atpX-TNF. Wherein thesaid expression cassette of TNF-α gene was located in the downstream ofboth promoters chlL and atpA, which can strengthen expression of theTNF-α gene.

[0071] 3. On the vector pUC-atpX-MD, there is the cloned expressioncassette of aadA gene, atpA5′-aadA-rbcL3′, which can expressaminoglycoside-3′-adenylate transferase (i.e. spectinomycin resistanceselectable marker). The vector pUC-atpX-MD was digested with EcoR V andSac I, then a 1.9 kb expression cassette of aadA gene was obtained. Thechloroplast intermediate expression vector p64C-atpX-TNF prepared instep 2 was filled to blunt ends using T₄ DNA polymerase after digestionwith Not I, subsequently, digested with Sacl, and finally inserted intop64C-atpX-TNF to construct the chloroplast expression vector p64C-TNF-MD(Fig. 1), which contained the expression cassette of human TNF-α gene(atpA5′-TNF-rbcL3′) and the expression cassette of aadA gene(atpA5′-aadA-rbcL3′). The expression cassette of TNF-α gene was directlylocated in the downstream of the chlL5′ promoter, which strengthens itsexpression under the control of the both promoters chlL and atpA.Furthermore, the expression cassette of aadA gene can expressspectinomycin resistance, which is helpful for screening therecombinants of Dunaliella Salina.

[0072] III. Introducing Foreign Target Genes into the Cells ofDunaliella Salina

[0073] 1. Introducing Foreign Target Genes into the Cells of DunaliellaSalina by Electroporation

[0074] The cells of Dunaliella Salina cultured in fluid medium for 5days were centrifuged at 1000 rpm for 15 min, and the supernatant wasdiscarded. After treatment with the solution containing 0.2M mannite and0.2M sorbitol, the cell density of Dunaliella Salina was adjusted to10⁸/ml by adding Buffer A (0.5 M CaCl₂ in 0.1 M Tris, final pH 7.2.).After the plasmid containing the foreign gene at a final concentrationof 10 μg/ml and salmon sperm DNA at a final concentration of 25 μg/mlwere mixed well, the mixture was placed on ice for 5-10 min, and then0.5 ml of cooled mixture was transferred into 0.2 cm electrode gapcuvette. The electroporation was performed at an electrical condition of2.6 KV/cm field strength, 200 ohms resistance, and 25 μF capacitance,corresponding to a time range of 5-10 msec, using a Gene Pulser IIsystem (Bio-Rad). Following delivery of the electrical pulse, 0.5 mlBuffer A was added to the suspension and kept on ice for 10 min, and the“clump” of cells was gently shaken to disperse into small pieces.Finally, the treated cells of Dunaliella Salina were cultured under theconditions suitable for their growth.

[0075] 2. Introducing Foreign Target Genes into the Cells of DunaliellaSalina with Gene Gun

[0076] The cells of Dunaliella salina cultured for 5 days in liquidmedium were centrifuged at 1000 rpm for 15 min. Subsequently, thesupernatant was discarded and the cell density of Dunaliella Salina wasadjusted to 10⁸/ml with Dunaliella Salina liquid medium, and then 0.5 mlof the cells was smeared in the center of the solid medium plate on acircle of about 3 cm in diameter and finally was blown dry underaspectic condition for further use.

[0077] For microprojectile bombardment, prewashed 50 μl aliquots of goldpowder suspension (60 μg/ml) were coated with 6 μg of plasmid containingthe foreign gene on ice. Fifty μl sterile aliquots of 2.5M CaCl₂ and 20μl of 0.1M spermidine were added to the above-mentioned mixturecontaining DNA particles, and mixed in a microfuge tube (1.5 ml). Theresulting mixture was vortexed for 3 min at room temperature andcentrifuged at 12000 rpm for 10 sec. After the supernatant was removed,the DNA particles were washed twice with 240 μl absolute ethanol andre-suspended in 60 μl absolute ethanol. Subsequently, 6-8 μl of thesuspension was spread onto the center of each macrocarrier andair-dried.

[0078] Bombardments were performed using the PDS-1000 helium-drivenbiolistics particle delivery system (Bio-Rad). Each plate was bombarded3 times at a rupture pressure of 600 psi, and then cultured under theconditions suitable for the growth of the cells of Dunaliella Salina.

[0079] 3. Introducing Foreign Target Genes into the Cells of Dunaliellasalina with PEG-Mediated Transformation

[0080] The protoplasts of Dunaliella Salina were prepared with 0.5 mlalga culture (cell density of 10⁷-10⁸/ml). After 1 μg of carrier DNA(calf thymus DNA) and 5-10 μg of plasmid DNA containing the foreign genewere mixed and gently swirled, the newly prepared suspension of theprotoplasts was incubated at room temperature for 20 min. One mlsolution of PEG (MW4000-6000) was added to the suspension of theprotoplast mentioned above, swirled, and then again incubated at roomtemperature for 10 min to boost the protoplast to take up DNA. Thetransformed protoplast was collected with centrifugation at 1000 rpm for15 min, re-suspended in 2 ml solution of 1 M sorbitol, and culturedunder the conditions suitable for the growth of Dunaliella Salina.

[0081] IV. Screening the Transformants of Dunliella Salina

[0082] The cells of Dunaliella Salina transformed with electroporation,gene gun or PEG were inoculated onto the solid media containing properscreening reagent(s). Under proper culture conditions, Dunaliella Salinacolonies would emerge 2-4 weeks later. These colonies were cultured inthe liquid media having no antibiotic for 3-5 days, and then screenedfor the second time on the solid media containing antibiotic. Afterbeing sub-cultured for more than 10 times, these transformants weregrowing luxuriantly.

Example 2

[0083] I. Dunaliella Salina Culture

[0084] The same culture used as described in example 1.

[0085] II. Construction of the Dunaliella Salina Expression PlasmidpCAMBIA-CtxB-SS1 Encoding Recombinant HbsAg (Fig. 2).

[0086] 1. Construction of the Recombinant HBsAg SS1 Fusion Gene

[0087] Plasmid pBS-SK-HBS contained sequences of S, Pre-S2 and Pre-S1genes of hepatitis B virus. Four primers for amplification of the geneslisted above were designed as follows: Primer 1: 5′-GCAGTCGACCCAATGGAGAGCAC-3′;        Sal I Primer 2: 5′-GCGGGTACCAGG AATGTATACCC-3′;       Kpn I Primer 3: 5′-CTGGGTACCCCA AATCCTCTGGG-3′;        Kpn IPrimer 4: 5′-GCGGCATGCTTA GTTGGGGTTG-3′;        Sph I

[0088] The S gene fragment encoding 1-226 amino residues of HBsAg andthe PreS1 gene fragment encoding 20-48 amino residues of PreS1Ag wereamplified using primers 1 and 2, and primers 3 and 4, respectively.After restriction enzyme digestion and recovery, the two fragments wereligated at the Sal I /Sph I site in the plasmid pUC18 to construct a newplasmid named pUC18-SS1, which contains the fusion gene SS1 encodingrecombinant hepatitis B surface antigen comprising 1-226 amino residuesof HBsAg and 20-48 amino residues of PreS1Ag.

[0089] 2. Construction of CtxB-SS1 Fusion Gene

[0090] (1) A pair of primers was designed according to the cholera toxinB subunit gene sequence reported. Forward primer: 5′-GCGGGATCCATGATTAAATTAAAATTTGG-3′;          BamH I Reverse primer: 5′-GCGGTCGACAGGATTTGCCATACTAATTGC-3′;         Sal I

[0091] When the genomic DNA of Vibrio cholerae (O139) was used as atemplate for PCR, a CtxB gene of 380 bp was obtained by PCRamplification, and then the CtxB gene was cloned into plasmid pUC18after digestion with BamH I/Sal I to construct a new plasmid pUC18-CtxB.

[0092] (2) A SS1 fusion gene of 770 bp in length was obtained afterdigestion of plasmid pUC18-SS1 with Sal I/Sph I, and then ligated at theSal I/Sph I site in plasmid pUC18-CtxB to construct plasmidpUC18-CtxB-SS1 which contains the CtxB-SS1 fusion gene consisting ofCtxB and SS1 genes.

[0093] 3. Construction of Hsp70B 5′-CtxB-SS1-T-Nos Expression Cassette

[0094] Plasmid pSP72-Hsp-Nos contained heat shock protein Hsp70B 5′promoter of Dunaliella Salina and T-Nos terminator. Both plasmidpSP72-Hsp-Nos and plasmid pUC18-CtxB-SS1 were digested with BamH I/SphI, and then the CtxB-SS1 fusion gene was ligated between the Hsp70B 5′promoter and the T-Nos terminator to construct a new plasmidpSP72-CtxB-SS1 containing an intact expression cassette in which theCtxB-SS1 fusion gene transcribes under the control of Hsp70B 5′promoter.

[0095] 4. Construction of Expression Plasmid pCAMBIA-CtxB-SS1 ofDunaliella Salina

[0096] There were two matrix attachment regions (MAR) with the sameorientation, MAR1 and MAR2, in Dunaliella Salina expression vectorpCAMBIA-DS1644 (Fig. 3). Between MAR1 and MAR2, the following elementswere linked in sequence:(a) the expression cassette of Nit5′-Nit1-T-Nos,which can express nitrate reductase used to screen the transgenicDunaliella Salina, (b) a multiple cloning site MCS derived from pUC18,and (c) the expression cassette of Nit5′-BAR-T-Nos, which expresses aPPT resistance used as an assistant screening. An intact expressioncassette of Hsp70B5′-CtxB-SS1-T-Nos was acquired by digestion of plasmidpSP72-CtxB-SS1 with EcoR I/Xho I and was ligated at EcoR I/Sal I site inplasmid pCAMBIA-DS1644 to construct expression plasmid pCAMBIA-CtxB-SS1(Fig. 4). Homologous recombination mediated by MAR1 and MAR2 can takeplace, which makes the expression cassettes, including Nit1, CtxB-SS1and BAR, integrated to the active transcription region of the DunaliellaSalina chromosome. After being screened by the PPT resistance and by thenitrate selective culture, the transformed Dunaliella Salina wouldhighly express the fusion protein of CtxB and SS1 induced by changes oftemperature.

[0097] III. Introducing Foreign Target Genes into the Cells ofDunaliella Salina

[0098] 1. Introducing Foreign Target Genes into the Cells of DunaliellaSalina with Electroporation

[0099] The same introduction as described in example 1.

[0100] 2. Introduction Foreign Target Genes into the Cells of DunaliellaSalina with Gene Gun

[0101] The same introduction as described in example 1.

[0102] 3. Introducing Foreign Target Genes into the Cells of DunaliellaSalina with PEG

[0103] The same introduction as described in example 1.

[0104] IV. Screening the Transformants of Dunaliella Salina

[0105] The cells of Dunaliella Salina transformed with electroporation,gene gun or PEG, etc., were washed with 1 ml culture fluid A containing5 mM NH₄Cl, 5 mM NaNO₃, cultured for 2-3 days with 300 lux, and theninoculated into culture fluid A containing 3 μg/ml PPT. After thetransformed cells were cultured for 5-7 days under the followingconditions: 12:12 light-dark cycle with 1600 lux, they were againinoculated into culture fluid B containing 10 mM NaNO₃ and cultured for7-10 days. At last, the cells were smeared onto the solid media thatwere made up of culture fluid B and agar, and continued to culture for10-15 days until colonies of the transformed Dunaliella Salina emerged.

1 6 1 23 DNA Artificial Sequence Description of Artificial SequenceSYNTHETIC PRIMER- SENSE; DERIVED FROM HBsAg 1 gcagtcgacc caatggagag cac23 2 23 DNA Artificial Sequence Description of Artificial SequenceSYNTHETIC PRIMER- SENSE; DERIVED FROM HBsAg 2 gcgggtacca ggaatgtata ccc23 3 23 DNA Artificial Sequence Description of Artificial SequenceSYNTHETIC PRIMER- SENSE; DERIVED FROM HBsAg 3 ctgggtaccc caaatcctct ggg23 4 22 DNA Artificial Sequence Description of Artificial SequenceSYNTHETIC PRIMER- SENSE; DERIVED FROM HBsAg 4 gcggcatgct tagttggggt tg22 5 29 DNA Artificial Sequence Description of Artificial SequenceSYNTHETIC PRIMER-SENSE; DERIVED FROM CHOLERA TOXIN B 5 gcgggatccatgattaaatt aaaatttgg 29 6 30 DNA Artificial Sequence Description ofArtificial Sequence SYNTHETIC PRIMER-ANTISENSE; DERIVED FROM CHOLERATOXIN B 6 gcggtcgaca ggatttgcca tactaattgc 30

What is claimed is:
 1. A transgenic Dunaliella Salina bioreactorcomprising a Dunaliella Salina as host, a foreign target gene and aselectable marker.
 2. A bioreactor as claimed in claim 1, wherein saidforeign target gene is derived from at least one selected from the groupconsisting of humans, animals, plants or microorganisms.
 3. A bioreactoras claimed in claim 2, wherein said foreign target gene derived frommicroorganisms and plants is at least one selected from the groupconsisting of HBsAg, measles virus antigen, foot-and-mouth disease virusantigen, rabies virus antigen, larvacide, cytokinin, endochitinase,glucose-amylase P, thaumatin, seed-stored protein genes, etc.
 4. Abioreactor as claimed in claim 2, wherein said foreign target genederived from humans and animals is at least one selected from the groupconsisting of angiostatin, endostatin, hematoglobin, human factor III,human erythropoietin interferon, obese protein, human interleukin, humangranulocyte colony stimulating factor, human macrophage colonystimulating factor, streptokinase, human protein kinase, growth hormone,tissue plasminogen activator, defensin, tumor necrosis factor, epidermalgrowth factor, bovine chymosin, and antibiotic peptide genes, etc.
 5. Abioreactor as claimed in claim 1, wherein said selectable marker is atleast one selected from the group consisting of spectinomycin orstreptomycin resistance encoded by aadA gene, chloromycetin resistanceencoded by cat gene, kanamycin or neomycin resistance encoded by npt IIor neo gene, hygromycin resistance encoded by hyg gene, herbicidephosphinothricin (PPT) resistance encoded by Bar gene, etc.
 6. A methodfor preparing the transgenic Dunaliella Salina bioreactor, furthercomprises the following steps: (a) introducing foreign target genes intothe cells of Dunaliella Salina using the transformation techniques. (b)screening transformed cells of Dunaliella Salina.
 7. A method as claimedin step (a) of claim 6, wherein said transformation techniques are oneor more of the methods for genetic transformation selected from thegroup consisting of biological, physical and/or chemical methods.
 8. Amethod as claimed in claim 7, wherein said biological method is a methodintroducing foreign target genes into Dunaliella Salina cells byagrobacterium Ti plasmid transformation system and/or plant virus vectorsystem.
 9. A method as claimed in claim 7, wherein said physical andchemical methods can be one or more of the group consisting of PEG,liposome, electroporation, ultrasonic delivery, gene-gun,microinjection, ultraviolet laser microbeam, glass bead agitation andaerosol gene delivery.
 10. A method as claimed in any one of claims 7 to9, further comprises the steps of constructing Dunaliella Salinaexpression vector and/or culturing Dunaliella Salina before introducingforeign target genes into the cells of Dunaliella Salina.
 11. The usesof any transgenic Dunaliella Salina bioreactor as claimed any one inclaims 1 to 6 in production of drugs or vaccines for humans and/oranimals, and phytohormones.